Sabine Leonhardt

Amino Acid Neurotransmitter Release in the Preoptic Area of Rats during the Positive Feedback Actions of Estradiol on LH Release

To investigate the role of amino acid neurotransmitters in the regulation of LH secretion in ovariectomized (ovx) rats with or without estrogen substitution, we measured the release rates of gamma-aminobutyric acid (GABA), taurine, glycine, aspartate, glutamate, homocysteic acid, and also of the neurally inactive amino acids serine and glutamine in push-pull perfusate samples of the preoptic/anterior hypothalamic area (PO/AH) collected at 30-min intervals. To achieve this we had to develop a highly sensitive assay utilizing phenylisothiocyanate prederivatization which was followed by HPLC chromatography. In confirmation of our earlier results we observed again a conspicuous drop of preoptic GABA release prior to and during the time of estrogen-induced LH surge. In addition, the release rates of the excitatory amino acid neurotransmitters aspartate and glutamate in the PO/AH increased during this time. Interestingly, also secretion of taurine and glycine was increased during the LH surge, whereas preoptic release rates of serine and glutamine and of homocysteic acid, the putative endogenous ligand of the so-called N-methyl-D-aspartate receptor, remained unchanged. No such changes of amino acid neurotransmitters release rates were observed in ovx rats. This finding underlines that the changes of amino acid secretion in ovx estrogen-primed rats are likely due to the influence of the steroid rather than due to a diurnal rhythm. We conclude that GnRH neurons are under a tonic inhibitory tone exerted by GABA which is relieved during the time of the estrogen-induced LH surge. During this time, aspartate and glutamate may have additional stimulatory effects on GnRH neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Gamma-Aminobutyric Acid Neurons in the Preoptic/Anterior Hypothalamic Area Synchronize the Phasic Activity of the Gonadotropin-Releasing Hormone Pulse Generator in Ovariectomized Rats

To achieve a bolus-type release of gonadotropin-releasing hormone (GnRH) into the portal vessels it is required that GnRH neurons exert phasic and synchronous activity. The activity of GnRH neurons appears to be under an inhibitory influence of the amino acid neurotransmitter gamma-aminobutyric acid (GABA). Preoptic GABA concentrations in ovariectomized (OVX) rats decrease prior to a luteinizing hormone (LH) episode. This reduction of GABAergic activity in the preoptic/anterior hypothalamic area (PO/AH) may be the synchronizing signal for the simultaneous release of GnRH from the hypothalamus. To further study the role of GABA in controlling the GnRH pulse generator we applied GABA, 3-mercaptopropionic acid (MPA) or bicuculline (BIC) locally into the PO/AH by means of push-pull cannulae (PPC). PPC were implanted into the PO/AH of OVX rats and the contralateral, not PPC-implanted PO/AH was lesioned electrochemically. The effects of GABA, MPA or BIC on the GnRH pulse generator were determined by measuring LH levels in blood samples collected in 5-min intervals. Local application of GABA into the PO/AH caused a pronounced reduction of average LH secretion and abolished LH pulsatility. This inhibitory effect was completely reversible. Results of intrapreoptic MPA application on GABA secretion were variable. In only 45% of treated rats MPA caused a reduction of GABA secretion which was associated with a cessation of pulsatile LH release. A pronounced reduction of LH secretion and pulsatility was observed upon local application of the GABA antagonist BIC. Based on these data we propose that oscillating GABA levels in the PO/AH may be the synchronizing signal which triggers bolus release of GnRH into the portal vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Preoptic rather than mediobasal hypothalamic amino acid neurotransmitter release regulates GnRH secretion during the estrogen-induced LH surge in the ovariectomized rat.

Inhibitory and excitatory amino acid neurotransmitters have been suggested to participate in the feedback actions of estradiol (E2) on LH secretion. In the rat estrogen-receptive neurons have been demonstrated in the preoptic/anterior hypothalamic area (POA) and mediobasal hypothalamus/median eminence (MBH) and many of these neurons utilize gamma-aminobutyric acid (GABA) as neurotransmitter. The actions of excitatory amino acids (EAA) differ in ovariectomized (ovx) and ovx E2-substituted rats indicating that EAAs also participate in the positive feedback action of E2 on LH release. However, little information is available as to whether in vivo these transmitters exert their effects in the POA, where most of the GnRH perikarya are located, or in the MBH, i.e. at the nerve terminals. Therefore we conducted push pull cannula perfusions to compare the release rates of GABA, aspartate (ASP) and glutamate (GLU) in the MBH and POA. A subcutaneous implant of a silastic tube containing E2 resulted in LH surges in the afternoon of all treated animals. Prior to and during this LH surge the MBH release rates of neither GABA nor ASP nor GLU were significantly altered. In contrast, a conspicuous drop in preoptic GABA release occurred prior to and during the time of estrogen-induced LH surges and this was accompanied by enhanced preoptic secretion of ASP and GLU. In conclusion, we present the first data about amino acid release in the MBH during the E2-induced LH surge. Since only in the POA the LH surge is associated with changes in amino acid release, it appears that both inhibitory and excitatory amino acids act at the level of the GnRH cell bodies and/or dendrites and not on GnRH nerve terminals to mediate the feedback mechanism of E2 on LH release.

The GABAergic Control of Gonadotropin-Releasing Hormone Secretion in Male Rats during Sexual Maturation Involves Effects on Hypothalamic Excitatory and Inhibitory Amino Acid Systems

In order to evaluate the possible participation of the hypothalamic excitatory and inhibitory amino acid neurotransmitter systems in the GnRH release response to GABAergic drugs, hypothalami (preoptic and mediobasal area) of immature (26 days of age) and adult male rats were perifused with GABA-A and -B agonists and antagonists. GnRH and amino acid neurotransmitter concentrations (glutamate, taurine, GABA) were measured in perfusate samples collected every 15 min during 150 min. In immature rats, muscimol and baclofen (GABA-A and GABA-B agonists, respectively) increased GnRH, glutamate and GABA release and decreased taurine output, while in adults these agonists showed opposite effects on GnRH and glutamate release, and increased GABA and taurine output. On the other hand, in immature rats bicuculline and phaclofen (GABA-A and GABA-B antagonists, respectively) decreased GnRH, glutamate and GABA release, increasing taurine outflow. In adult animals, these antagonists enhanced GnRH and glutamate release, decreasing taurine and GABA outflow. These results indicate that GABA stimulates GnRH release in immature male rats and confirm the inhibitory role of this amino acid neurotransmitter in adult animals. This effect might be associated, at least partially, with the modifications observed in the excitatory and inhibitory amino acid release. On the other hand, in immature rats, stimulation of GABA-A and GABA-B receptors increased GABA release. Although ultrastructural studies have not produced any evidence of GABA-GABA neurointeractions, our results suggest the existence of a positive feedback mechanism of GABA autoregulation active during the prepubertal stage. Participation of this mechanism in the onset of puberty cannot be discarded.

Activation of Central GABAA- but Not of GABAB – Receptors Rapidly Reduces Pituitary LH Release and GnRH Gene Expression in the Preoptic/Anterior Hypothalamic Area of Ovariectomized Rats

gamma-Aminobutyric acid (GABA) exerts an inhibitory action on gonadotropin-releasing hormone (GnRH) release from the hypothalamus. In vivo, this inhibitory action appears to be mediated via the GABAA receptor since in ovariectomized (ovx) rats and sheep direct application of muscimol (MUS), a GABAA agonist, into the preoptic area (POA), the site were the GnRH cell bodies are located, caused an immediate reduction of LH release. This effect may be the result of an inhibition of GnRH release but also GnRH biosynthesis may be affected. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) we now addressed the question, whether an acute inhibition of the GnRH pulse generator in ovx rats by GABA involves reduction of GnRH biosynthesis as determined by GnRH mRNA levels in micropunches of the POA. To activate either the GABAA or GABAB receptor, we injected intraventricularly (icv) MUS or baclofen (BAC). Intracerebroventricular injection of 10 nmol MUS caused a rapid and lasting inhibition of LH release from about 7.5 ng/ml (pretreatment value) to approximately 1.5 ng/ml. Neither application of BAC or saline (control injections) affected LH secretion. Two hours after icv injections, rats were decapitated and GnRH mRNA levels were determined. MUS induced a pronounced decrease of GnRH levels in the POA (control rats: 2.26 pg GnRH mRNA; MUS-treated rats: 0.85 pg, n = 10/group). BAC was without any effect on GnRH mRNA levels. Thus, we confirm the inhibitory action of GABA on LH release in vivo which is exerted via the A-subtype of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats

Pituitary adenylate cyclase activating polypeptide (PACAP) is produced by hypothalamic neurons which terminate within the median eminence suggesting that it may be a hypophysiotropic hormone. However, little endocrine activity has been ascribed to the peptide. Therefore we studied the effects of PACAP on prolactin (Prl) release from dispersed cultivated rat pituitary cells in vitro using conventional cultures as well as the reverse hemolytic plaque assay (RHPA). Furthermore the effects of the peptide on in vitro GH release were assessed. In addition, the activity of the peptide on in vivo release of Prl and GH was studied in hypothalamus-lesioned animals. PACAP dose dependently inhibited Prl release form dispersed pituitary cells in both, monolayer cell cultures and the RHPA, whereas GH secretion was not affected. In hypothalamus-lesioned rats which have high Prl levels due to the absence of hypothalamic dopamine, PACAP further stimulated Prl release. Serum GH increased more than 20 fold in response to the intravenous PACAP infusion. Thus in vitro (inhibition of Prl release, no effect on GH release) and in vivo (stimulation of both hormones) experiments yielded contradicting effects of PACAP on pituitary hormone release. We suggest that PACAP may stimulate the release of a paracrine, yet unknown factor which in the intact pituitary overrides the direct inhibitory action of PACAP on the lactotropes. The same or another paracrine factor may also enhance in vivo GH release. In cell culture the paracrine factor is diluted by the medium. Therefore the peptide never reaches effective concentrations which are present within the intact pituitary tissue.

A Norepinephrine-Dependent Mechanism in the Preoptic/Anterior Hypothalamic Area but Not in the Mediobasal Hypothalamus Is Involved in the Regulation of the Gonadotropin-Releasing Hormone Pulse Generator in Ovariectomized Rats

Pulsatile gonadotropin secretion from the pituitary is dependent upon the gonadotropin-releasing hormone (Gn-RH) pulse generator producing intermittent release of the neuropeptide into the portal vessels. Various neurotransmitters seem to be involved in the regulation of pulsatile Gn-RH release. The present study was an attempt to determine in vivo the temporal relation of preoptic/anterior hypothalamic area (PO/AH) norepinephrine (NE) release and pulsatile luteinizing hormone (LH) secretion in ovariectomized rats. To assess whether NE acts in the PO/AH to maintain pulsatile Gn-RH release, we applied locally an alpha 1-receptor antagonist into this structure. Push-pull cannulae (PPC) were implanted into the PO/AH of ovariectomized rats. The contralateral, not PPC-implanted PO/AH was lesioned electrochemically. Another group of ovariectomized rats was implanted with a PPC into the mediobasal hypothalamus. Two experiments were performed: (1) To determine whether the PO/AH or the mediobasal hypothalamus is the site where NE exerts its stimulatory effect on LH secretion, we applied doxazosine, a new specific alpha 1-receptor antagonist, locally into these structures by means of PPC. The effect of this adrenergic drug on the Gn-RH pulse generator was examined by measuring blood LH levels. (2) To study the temporal relation between in vivo release rates of NE and amine metabolites in the preoptic area and pulsatile pituitary LH secretion, preoptic perfusates and blood samples were collected at 5-min intervals. Brain perfusates were subjected to high-performance liquid chromatography-electrochemical analysis. In blood samples LH concentrations were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol Modulates the LH Release Response to N-Methyl-D-Aspartate in Adult Female Rats: Studies on Hypothalamic Luteinizing Hormone-Releasing Hormone and Neurotransmitter Release

We investigated the effect of ovariectomy (OVX) and subsequent estradiol benzoate (EB) treatment upon the N-methyl-D-aspartate (NMDA)-induced LH secretion in adult female rats. Furthermore, the release of LHRH, norepinephrine (NE), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA) and gamma-aminobutyric acid (GABA) from superfused hypothalamic fragments explanted from OVX and OVX-EB rats was determined. Two weeks after OVX, animals received EB (100 mg/kg) s.c., or oil vehicle (OVX-EB or OVX groups, respectively). Two days thereafter, at 09.00 h, NMDA (15 or 30 mg/kg) was given as an i.v. bolus; blood samples were drawn before and 10 min after drug administration. In OVX rats, NMDA had no significant effect on LH levels, whereas it stimulated LH release in OVX-EB animals at both doses tested (315 and 362% from basal values, p < 0.001). For hypothalamic superfusion studies OVX and OVX-EB animals were decapitated at 09.00 h, and the mediobasal hypothalami (MBH) dissected on ice. NMDA (10(-4) M) was added to the superfusion medium for a 10 min period. Basal LHRH release (OVX: 1.41 +/- 0.18; OVX-EB: 1.59 +/- 0.28 pg/10 min/MBH) was significantly (p < 0.05) enhanced by NMDA (OVX: 2.97 +/- 0.95; OVX-EB: 2.80 +/- 0.61 pg/10 min/MBH). EB treatment had no significant effect on basal or NMDA-induced LHRH output.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative analysis of different puberty inhibiting mechanisms of two GnRH agonists and the GnRH antagonist cetrorelix using a female rat model.

GnRH agonists are the established treatment of precocious puberty caused by premature stimulation of gonadotropin secretion. It has been reported that after an initial stimulation (“flare-up”) they reduce LH secretion by desensitization of pituitary GnRH receptors. Little has been published about the use of GnRH antagonists such as cetrorelix to control the onset of puberty and whether they are potentially advantageous compared with GnRH agonists. We conducted two multigroup experiments (12 and 10 d, respectively) treating prepubertal/peripubertal female rats with either the GnRH agonist triptorelin or buserelin and compared them with rats treated with the GnRH antagonist cetrorelix and controls to assess the effects on pubertal progress and serum hormones. In the second experiment, the effects of buserelin and cetrorelix on gene expression of the GnRH receptor, LH-β, FSH-β, and the alpha subunit genes in the pituitary were also investigated. Cetrorelix, triptorelin, and buserelin retarded the onset of puberty as determined by delayed vaginal opening, lower ovarian weights, and lower serum estradiol levels. However, although LH and FSH levels were stimulated by both agonists, they were inhibited by cetrorelix. In the cetrorelix versus buserelin experiment, pituitary gene expression of the GnRH receptor and LH-β subunit were significantly lower in cetrorelix treated rats compared with controls whereas buserelin had little effect. Expression of FSH-β and alpha subunit were stimulated by buserelin but not by cetrorelix. Even though all three of these GnRH analogues inhibited gonadal development and delayed the onset of puberty , the GnRH agonists had stimulating and inhibiting effects on the pituitary-gonadal axis whereas cetrorelix exerted only inhibiting effects. We conclude from this female rat model that cetrorelix may offer advantages for a more controlled medical treatment of precocious puberty compared with GnRH agonist treatment.

Cranial irradiation of female rats causes dose-dependent and age-dependent activation or inhibition of pubertal development.

Cranial irradiation in prepubertal children with leukemia or brain tumors can lead to precocious or in high doses to late puberty. To unravel the underlying mechanisms, we developed a rat model with selective cranial Co60-irradiation technique. Infantile (12–16 d old) or juvenile (21–23 d old) female Sprague-Dawley rats received a single dose of 4, 5, 6, 9 or 2 × 9 Gy (at days 21 and 23). Each group consisted of 7–20 animals. High radiation doses (9 Gy and more) caused retardation of sexual development, whereas low radiation doses (5 or 6 Gy) led to accelerated onset of puberty in 20% of infantile irradiated rats animals as determined by vaginal opening. Interestingly, at peripubertal age (postnatal day 32–34), 5 or 6 Gy infantile irradiated rats had significantly higher serum LH levels stimulated by GnRH and estradiol levels (p < 0.05). 2 × 9 Gy irradiated rats had at the age of 3 mo a marked growth retardation and significantly lower GH levels than the controls (p < 0.05) whereas prolactin, FSH, TSH, T4, and corticosterone levels were comparable with controls. These studies demonstrate that the GnRH-pulse generator is very radiosensitive as precocious activation occurred after low dose irradiation (5 or 6 Gy) of infantile rats without any other endocrine disorder. High radiation doses (9 or 2 × 9 Gy) induced retardation of sexual maturation and later on growth hormone deficiency. Moreover this model of cranial irradiation seems to be suitable to study the molecular mechanisms of radiation induced pubertal changes.